The ability to degrade mRNA and re-use its component nucleotides is essential to viability of all living cells. There is increasing evidence that mRNA decay is a coordinated multistep process that is dynamically regulated in response to changes in cell physiology. However, limited information currently is available about the features of ribonucleases that determine their mode of action, their relative activities, and their response to physiological events. Similarly there is incomplete knowledge of the proteins and non-coding RNAs that can interact with ribonucleases and/or their substrates to modulate ribonucleolytic activity and specificity. The long-term objective of the proposed research is to achieve a better understanding of the mechanisms that regulate RNA decay in bacteria. The specific aims for the next project period are: 1) to elucidate the structural features of the RNase E and RNase G proteins that determine their specificity and mode of action, 2) to identify and investigate the effects of other cellular proteins that interact with these ribonucleases, regulate their actions, and allow them to respond to changes in cell physiology, and 3) to identify transcripts whose degradation by these ribonucleases is differentially affected by physiological changes, identify the regulatory proteins and/or small RNAs that mediate these effects, and elucidate the mechanisms of action of selected regulators. The research is multifaceted and will employ a combination of genetic and biochemical approaches--including mutational analysis of ribonuclease structure/function relationships, microarray-based investigations of changes in RNA decay in response to physiological events, protein purification and analysis, and biochemical study of the effects of regulator proteins and/or RNAs on ribonuclease action. While these investigations are intended to elucidate fundamental aspects of bacterial RNA decay, the information obtained is likely to facilitate the more efficient use of microorganisms to express biologically and medically important proteins and also to further the development of novel antimicrobial drugs that target RNA decay mechanisms in bacteria. [unreadable] [unreadable]